Homeostatic control of adiposity depends on the ability of the brain to sense and respond to changes in circulating indicators of body energy stores. This proposal is focused on understanding the molecular and cellular mechanisms by which two groups of neurons in the arcuate nucleus of the hypothalamus, those marked by expression of Agouti-related protein (Agrp) and Preproopiomelanocortin (Pomc), measure and respond to changes in leptin, insulin, and ghrelin. Our approach has been made possible by recent advances in transgenic technology and molecular imaging, and is based on the development of tools that allow us to control gene expression specifically in the Agrp/Npy and Pomc/Cart neurons. Using homologous recombination of bacterial artificial chromosome (BAC) clones in E. coli followed by transgenic introduction of the modified clones into mice, we have placed Cre recombinase under control of regulatory elements for the Agrp or Pomc genes. These Tg.AgrpCre and Tg.PomcCre mice will be applied to better understand how peripheral signals of body fuel stores and meal patterning are perceived and integrated by hypothalamic neurons by constructing and analyzing the phenotype of mice that are deficient for Stat3 or for Irs2 in either Agrp neurons, Pomc neurons, or both. We will also use a newly developed assay for dynamic measurement of phosphoinositide 3-kinase signaling in individual cells to determine how each subpopulation of arcuate nucleus neurons responds to leptin, insulin, or ghrelin in a brain slice preparation. Finally, we will determine if and how agents that increase long chain fatty acyl CoA levels intersect with Stag and PI3K signaling in Agrp and Pomc neurons. Together, these studies will provide fundamental new insight into cellular mechanisms governing the response of key hypothalamic neurons to hormonal and nutrient-related signals.